1. Field of the Invention
This invention relates to a tunable solid state laser system designed for matrix-assisted laser desorption/ionization (MALDI) applications, for example, large bio-molecules and DNA analysis.
2. Discussion of the Background
Mass spectrometric (MS) analysis of large biologically important molecules has made significant advances over the past several years. Increasingly, mild desorption methods have made possible the analysis of progressively larger macromolecules with minimal fragmentation, allowing the analysis of complex biological mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was first introduced by Karas, M.; Hillenkamp, F. Anal. Chem. 1988, 60, 2299, the entire content of which is hereby incorporated by reference.
MALDI-MS is now increasingly applied to the study of peptides, proteins and other bio-molecules for amino acid sequencing, structural analysis, modifications and non-covalent interactions. In comparison to the rapid development of MALDI for peptide and protein analysis, MALDI work on nucleic acids has been somewhat slower. This is due, in part, to the polyelectrolytic nature of nucleic acids, which combines well with most matrices and requires a relatively large deposition of energy to affect desorption of the macro molecules with an attendant risk of increased fragmentation. Recently, considerable attention has been directed to overcome these difficulties because of the great benefits which reliable, routine mass spectrometric techniques would offer in the field of molecular biology, and especially in the areas of gene analysis, identity testing, forensics, diagnosis of genetic disorders, and DNA sequencing.
Although MALDI-MS holds great potential for the analysis of DNA fragments with increased throughput and minimum sample degradation, it is presently limited mainly by the low ionization efficiency and fragmentation problems. MALDI-MS is further limited by the restricted selection of matrices that are accessible with fixed wavelength lasers.
Many of these limitations are a consequence of the current reliance on ultra violet (UV) lasers such as Nitrogen (337 nm), or frequency tripled, or quadrupled Nd:YAG (355 nm or 266 nm) lasers for MALDI. Although hundreds of potential matrix materials exist, only a limited number of useful matrices (approximately 30) are suitable for use with the conventional fixed wavelength lasers. The most successful matrix materials for nucleic acids in the UV range are picolinic acid and its derivatives, whereas succinic acid works well in the near IR region as demonstrated by Nordhoff et al, Nucl. Acids Res. 1994, 22, 2460, the entire content of which is hereby incorporated by reference. On the other hand, infrared IR laser sources can overcome the difficulties of molecular fragmentation, and also permit the use of a wide variety of laser desorption matrices, as shown by Cramer et al, J. Am. Soc. Mass Spectrom. 1996, 7, 1187, the entire content of which is hereby incorporated by reference. In studies, the IR MALDI resolution was found to be higher, and the sample molecules were stable during IR radiation, while the stability of the same sample molecules degraded under UV radiation. See Siegel et al, Anal. Chem. 1997, 69, 2716-2726, the entire content of which is hereby incorporated by reference.
CO2 (10.6 xcexcm) lasers have been used effectively since 1983 for laser desorption studies. Both fixed wavelength lasers such as Er:YAG (2.94 xcexcm) and CO2 and tunable sources such as free electron lasers (typically 2.2 to 7.0 xcexcm) and very recently tunable (2 to 3.5 xcexcm) Optical Parametric Oscillators (OPO) including Cr:LiSAF pumped KTA OPO have been utilized in a limited number of IR MALDI experiments. However, the high cost along with the complexity and size of these IR lasers, especially the free-electron lasers, have historically limited the use of IR lasers to only a few selected research groups. Furthermore, the commonly used OPO pumped by a Nd:YAG laser, which can generate IR output in the 2.7-3.2 xcexcm wavelength range, is cumbersome, expensive and requires expertise to operate and are thus mainly suitable for research facilities.
An examination of the literature reveals that the IR MALDI technique is not nearly as mature as the UV MALDI, and a great deal of work remains to be done before its full potential is realized. For example, the mass resolution obtained using a conventional IR-MALDI system is less than satisfying. Also, the influence of sample preparation and laser wavelength on the resulting mass spectra is significant.
Most time-of-flight (TOF) mass spectrometers are large, expensive instruments requiring considerable expenditure of time and personnel for their routine operation. This expense and expenditure has motivated the development of miniaturized, low cost TOF mass spectrometers that could have sufficient sensitivity and resolution to be utilized for all the comparative sequencing applications mentioned earlier. Taking advantage of the high time-resolution of current digitizers and using reflectrons to improve mass resolution, a miniature endcap TOF MS was recently demonstrated as being capable of providing structural information for biological molecules. See Cornish et al, in Proceedings of the 45th ASMS Conference on Mass Spectrometry and Allied Topics, 1997, the entire content of which is hereby incorporated by reference.
Although, in general, detection of oligomers with approximately 30 nucleotide bases (primers plus extensions) is required for most of the hybridization schemes that are being considered now, one object of the present invention is to permit facile, high sensitivity detection and resolution of oligomers up to 100-mer. The latter is indeed difficult utilizing current UV laser-based MALDI instruments, but utilization of the IR laser at optimal wavelengths as provided by the present invention is expected to greatly improve the signal to noise ratio in that range.
Thus, a widely tunable, compact, solid state IR (infrared) laser source covering a wide range of wavelengths from 2 to 5 xcexcm is critically needed for MALDI applications in order to expand the capabilities of MALDI MS to the range of IR wavelengths, where a large number of potential matrices exist.
Accordingly, one object of this invention is to provide an innovative, variable IR wavelength, miniaturized laser system to be used with a mass spectrometer, for example, a miniaturized endcap reflectron time-of-flight mass spectrometer (wich corresponds to a special mass spectrometer design known to those skilled in the art). The present invention provides higher sensitivity, higher resolution and higher mass accuracy measurements in MALDI applications. In addition, the laser system of the present invention furnishes the ability to select new amenable matrices and ultimately can provide analytical capabilities on a compact, user friendly, and inexpensive instrument that can be utilized in a variety of analytical, medical, environmental, and forensic settings. The laser system can also be applied to other MALDI configurations that include, general time of flight MS and ion trap MS.
Another object of the present invention is to provide a tunable infrared laser system that provides an infrared laser wavelength at 2-5 xcexcm range with a pulse energy up to a few mJ, thus providing a desorption and ionization source for the MALDI mass spectrometers.
Another object of the present invention is to provide an infrared laser system including a tunable diode pumped solid state (DPSS) laser and associated quasi-phase matched optical parametric oscillator (QPM OPO) or other nonlinear crystal, whereby a wide tuning is accomplished by tuning the pump laser frequencies without resetting the OPO crystal parameters.
Another object of the invention is to provide a tunable DPSS laser system based on Yb:YAG crystal which is effectively pumped by a semiconductor diode laser array(s) through an end-pumping or a side-pumping geometry to provide high quality beam laser pulses for pumping the OPO to generate stable IR laser pulses.
Another object of the invention is to provide a technique to convert the pump laser wavelength to an infrared wavelength by using a quasi-phase-matching nonlinear optical material such as PPLN, PPRTA, or other infrared laser material.
Another object of the invention is to provide a small, diode-pumped widely tunable, high repetition rate, laser source which is critically needed for a robust low cost IR-MALDI-MS instrument.
It is yet another object of the invention to provide interface optics designed for, and a method of, delivering the infrared laser pulse to the bio-sample inside the MS with an optimum efficiency and focusing for the maximum energy density at an optimum (controllable) size for best ionization efficiency and sensitivity of the MALDI/MS.
Another object of the present invention is to provide a novel Light Detection And Ranging (LIDAR) system including a novel infrared laser system.
These and other objects are achieved according to the present invention by providing a laser system and methods which integrate a DPSS tunable laser and OPO in a compact system, thereby generating an intermediate energy tunable near infrared laser pulse using a Yb:YAG laser crystal, tuning the output wavelength using a birefringent tuner BRT in a laser cavity, pumping the OPO with the output of this laser cavity to convert from a wavelength in the near infrared to a wavelength in the infrared region, and amplifying the tuning range by the OPO cavity from 2-5 xcexcm without angular rotation or temperature tuning of the OPO crystal. Output from the laser system can be focused onto samples for MALDI-MS analysis.
The entire laser system is integrated into a compact package, has low power consumption through an efficient diode pumping design, and provides stable laser output due to the reduced thermal loading of the laser system. This configuration consequently provides a long lifetime for the laser operation and robust performance that does not require intense maintenance and highly skilled operators.
The output of the above described laser/OPO system provides medium pulse energy, up to a few mJ, for example, 1-10 mJ, with relatively short pulses (up to 40 nsec) at a relatively high pulse repetition frequency PRF, for example, 2-100 Hz, or up to 1 kHz, if required. The compact design and low thermal dissipation from the efficient diode pumping design combined with the robust optomechanical design provides a high level of laser energy output stability (less than 3% fluctuation), which is a desirable feature for MALDI applications to assure high sensitivity and resolution. The output laser beam produces a well defined Gaussian profile which enables tight focusing at the sample location in the MS. The threshold for MALDI ionization is thus significantly reduced and sample consumption is minimized for economic sample analysis and improving the sensitivity. Combining the flexibility of focusing of the Gaussian laser beam with the wavelength tunability in the infrared region, this system provides a unique feature for MALDI analysis of large mass biomolecules including DNA and RNA, which has not been previously possible.